The long term goal of this proposal is to advance the understanding of the functional dynamics of the neurotransmitter:sodium symporter (NSS) family of transporters. NSS members including human dopamine, serotonin, and norepinephrine transporters harness sodium and chloride gradients to facilitate reuptake of neurotransmitters from the synapse into presynaptic neurons and glia. This function is vital for terminating neurochemical signals, maintaining intracellular neurotransmitter concentrations, and priming the cell for subsequent signaling events. NSSs participate in the regulation of mood, reward, and locomotion and have been implicated in diseases of the central nervous system such as depression, anxiety, autism, epilepsy, attention deficit hyperactivity disorder, and obsessive compulsive disorder as well as abuse and addiction to illicit drugs such as cocaine and amphetamine. Investigations in this proposal will focus on the leucine transporter (LeuT), a bacterial member of the NSS family and the preeminent model for understanding NSS function. While previous studies have begun to elucidate the structure and dynamics of LeuT, key questions related to the conformational cycle remain unanswered. The specific aims of this proposal will address the most critical of these questions: What helical motions are required to generate an intracellular permeation pathway? Where is this pathway located? What is the mechanistically relevant open-in conformation of LeuT? Answering these questions will be fundamental to understanding the transport mechanism of LeuT, which remains controversial. The design of these aims will test the hypothesis, supported by the hydrantoin transporter (Mhp1) family of structures, that LeuT functions by a rocker switch mechanism. This mechanism implies a rigid body reorientation of the pseudo-dimer motifs of LeuT that allows access to the primary substrate binding site from either side of the membrane. To address these questions, electron paramagnetic resonance (EPR) spectroscopy and computational modeling will be employed to define the nature and amplitude of conformational changes involved in LeuT transport. Site-directed spin labeling (SDSL) and EPR provide powerful tools to describe global dynamic motion as well as changes in residue environments indicative of local conformational changes. Furthermore, EPR measurements will be employed to generate the first empirical model of the open-in conformation using the de novo protein folding algorithm Rosetta. The work proposed here will provide novel insights into the conformational dynamics of LeuT and NSS transport. Furthermore, application of the hybrid technique of EPR spectroscopy and computational structure determination proposed in this work will represent a significant methodological advance in the field of membrane protein structure determination.